Human cancers arise through a series of genetic changes that transform normal cells into malignant tumors. Many of these changes are caused by genomic rearrangements and other errors during replication. To prevent such replication errors, cells have evolved DNA damage checkpoints, a sophisticated set of DNA quality control mechanisms. Central among them is the S-phase DNA damage checkpoint, a mechanism that slows replication in response to DNA damage. Genetic evidence in humans and mice suggest that the S-phase DNA damage checkpoint is crucial for preventing cancer; human patients with mutations that disrupt this checkpoint are prone to a variety of early-onset malignancies. Understanding the checkpoint's mechanism is essential for understanding the etiology of these cancers, and will fundamentally affect the way subsequent studies of this checkpoint are approached. The checkpoint has two branches: one that regulates the activation of replication origins and one that regulates the progression of replication forks. The mechanism of the fork-regulation branch of the checkpoint is not understood. Furthermore, the relative importance of the two branches in maintaining genomic stability is unknown. The proposed experiments are designed to i) to directly determine the extent to which regulation of origin firing and fork progression contribute to the slowing of replication in response to DNA damage, ii) to test the hypothesis that the fork branch acts to induce replication-coupled recombination and iii) to measure the relative contributions of the two branches to the maintenance of genomic stability. These experiments will take advantage of the fission yeast Schizosaccharomyces pombe as a model system. The conservation of checkpoints between fission yeast and humans makes fission yeast an excellent model for investigating these vital DNA damage surveillance pathways. The powerful genetic and biochemical tools available for fission yeast make it possible to rapidly identify key pathway members and rigorously test hypotheses about their functions. Understanding the fission yeast S-phase DNA damage checkpoint will provide an important framework for understanding how the human checkpoint maintains genomic stability. This understanding will lead to new therapeutic targets and diagnostic tools for the treatment and prevention of human cancer. PUBLIC HEALTH RELEVANCE: Many of the genetic changes that lead to cancer are caused by errors during DNA replication. Cells use the S-phase DNA damage checkpoint to prevent and repair such replication errors. The proposed research will elucidate the function of this checkpoint, allowing for the identification of new therapeutic targets and diagnostic tools for the treatment and prevention of human cancer.